Characterizing RNA-metal binding by Raman spectroscopy

通过拉曼光谱表征 RNA-金属结合

基本信息

批准号：
7796815
负责人：
PAUL R CAREY
金额：
$ 30.56万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-04-01 至 2013-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7796815
关键词：
7SL RNA Active Sites Adenine Bacteriophages Binding Binding Sites Biological Process Catalysis Catalytic RNA Cations Cell Nucleus Cells Chemicals Chemistry Clinic Clinical Cobalt Collaborations Complex Crystallography Cytokine Inducible SH2-Containing Protein Cytoplasm DNA Data Dependency Disease Drug Design Electrostatics Endoplasmic Reticulum Enzymes Foundations Frequencies Functional RNA Genetic Code Goals Guanine Guanosine Heart Hepatitis Delta Virus Imidazole Individual Introns Ions Isotopes Knowledge Label Laboratories Lasers Lead Libraries Life Link Location Magnesium Measures Membrane Messenger RNA Metals Methenamine Methods Microscope Microscopy Modality Modeling Molecular Conformation Monitor Mutate Mutation Nature Nucleotides Phase Phenylalanine-Specific tRNA Play Population Positioning Attribute Process Property Protein Biosynthesis RNA RNA Binding RNA Probes RNA Splicing Raman Spectrum Analysis Reaction Regulation Relative (related person)Research Research Technics Resolution Ribosomal RNA Ribosomes Roentgen Rays Role Running Samarium Signal Recognition Particle Signal Transduction Site Small Nuclear RNA Small RNA Solutions Spectrum Analysis Stretching Structure Surface System Techniques Testing Time Titrations Translating Vertebral column Work X-Ray Crystallography analog base catalyst chemical reaction chemical synthesis enzyme mechanism functional group gene function group I ribozyme inorganic phosphate insight light scattering mRNA Precursor novel novel strategies phenylalanine-tRNA phosphodiester phosphorothioate programs protonation public health relevance research study scaffold shape analysis three dimensional structure tool

项目摘要

DESCRIPTION (provided by applicant): RNA participates in a wide range of biological processes. In addition to its role in information transfer in the genetic code, it can act as a structural scaffold, a catalytic enzyme, or a regulatory signal. In the clinical context it is both a target for drug design and a tool for regulating gene function. Much of RNA function is driven by its interaction with metal ions and metals play a pivotal role in maintaining RNA's structure and promoting the catalytic mechanism. The principle goals of this proposal are to develop new modalities to characterize metal RNA interactions and the chemical reactions they promote. The methods involve the use of Raman spectroscopy to probe RNA in solution and Raman microscopy to study reactions in single crystals of RNA. The solution studies will use small RNA fragments, less than 40 nucleotides, in order to characterize a metal cation binding at a single phosphate group along with conformational changes in the adjacent phosphodiester backbone. The single crystal studies center on four RNAs, the Hepatitis Delta Virus (HDV) ribozyme, the P4-P6 domain and Twort ribozyme from group I introns, and phenylalanine tRNA. Magnesium binding sites in the crystal structures that have been characterized by X-ray crystallography are being used to provide the Raman signatures of -PO2- groups making inner sphere contacts with magnesium. The low frequency Raman spectral region provides the spectra, and thus chemical information, associated with the bound metals themselves. A major theme is to use Raman crystallography, combined with X-ray crystallography to provide unambiguous answers to outstanding issues of mechanism. For example, for HDV crystals we label active site G1and U-1 groups with 13C and 15N isotopes to allow us to follow these residues as catalytic parameters such as pH and Mg concentration are changed. These observations will be extended by using the labeling approach to define the chemistry of the key active site groups as the chemical reaction is being carried out in the crystal. An overarching aspect of this proposal is its interdisciplinary nature that involves collaborators at three locations. The Raman spectroscopy and Raman crystallography are undertaken in the Carey laboratory at CWRU, while the expertise and material for solution studies is provided by Drs. Mike Harris and Eric Christian from CWRU and Dr. Kwaku Dayie at the Cleveland Clinic. The work on RNA crystals is based on collaborations with Dr. Phil Bevilacqua at Penn State and Dr. Barbara Golden at Purdue. PUBLIC HEALTH RELEVANCE: Although RNA is a close relative of DNA it is far more versatile and plays many roles in the life of the cell. Thus, an understanding of the properties of RNA lies at the heart of our knowledge of the processes of life and to be able to control them when malfunction leads to disease states. The present proposal sets out to determine how RNA acts as a catalyst to bring about chemical reactions. The research involves following chemical reactions in very small RNA crystals using a laser light scattering technique.

描述（由申请人提供）： RNA参与了广泛的生物学过程。除了其在遗传密码中的信息传递中的作用外，它还可以充当结构支架，催化酶或调节信号。在临床背景下，它既是药物设计的目标，又是调节基因功能的工具。 RNA功能的大部分与金属离子的相互作用驱动，金属在维持RNA的结构和促进催化机制方面起着关键作用。该提案的主要目标是开发新的方式来表征金属RNA相互作用及其促进的化学反应。该方法涉及将拉曼光谱法用于探测溶液中的RNA和拉曼显微镜在RNA的单晶中研究反应。溶液研究将使用小于40个核苷酸的小RNA片段，以表征单个磷酸基团的金属阳离子结合以及相邻磷酸二酯主链的构象变化。单晶研究集中在四个RNA，肝炎三角病病毒（HDV）核酶，I组内含子的P4-P6结构域和Twort核酶和苯丙氨酸TRNA上。已经使用X射线晶体学表征的晶体结构中的镁结合位点用于提供-po2-组的拉曼特征，从而使内部球体与镁接触。低频拉曼光谱区域提供了与结合金属本身相关的光谱，从而提供了化学信息。一个主要主题是使用拉曼晶体学，并结合X射线晶体学，为出色的机制问题提供明确的答案。例如，对于HDV晶体，我们将活性位点G1and U-1组标记为13C和15N同位素，以使我们能够遵循这些残基，因为催化参数（例如pH和MG浓度）。这些观察结果将通过使用标记方法来扩展，以定义关键活性位点基团的化学性质，因为在晶体中进行了化学反应。该提案的一个总体方面是其跨学科性质，涉及三个地点的合作者。拉曼光谱和拉曼晶体学是在CWRU的Carey实验室进行的，而溶液研究的专业知识和材料由DRS提供。 CWRU的Mike Harris和Eric Christian和Cleveland诊所的Kwaku Dayie博士。 RNA晶体的工作是基于与宾夕法尼亚州立大学的Phil Bevilacqua博士和Purdue的Barbara Golden博士的合作。公共卫生相关性：尽管RNA是DNA的近亲，但它的用途广泛得多，并且在细胞的生活中起着许多作用。因此，对RNA的特性的理解是我们对生命过程的了解的核心，并在出现故障导致疾病状态时能够控制它们。目前的提案旨在确定RNA如何充当引起化学反应的催化剂。该研究涉及使用激光光散射技术在非常小的RNA晶体中进行化学反应。